U.S. patent application number 14/991889 was filed with the patent office on 2016-07-14 for nanofibrous photoclickable hydrogel microarrays.
The applicant listed for this patent is The Regents of the University of Colorado, a body corporate. Invention is credited to Stephanie Bryant, Michael Floren, Sadhana Sharma, Wei Tan.
Application Number | 20160202241 14/991889 |
Document ID | / |
Family ID | 56367377 |
Filed Date | 2016-07-14 |
United States Patent
Application |
20160202241 |
Kind Code |
A1 |
Floren; Michael ; et
al. |
July 14, 2016 |
NANOFIBROUS PHOTOCLICKABLE HYDROGEL MICROARRAYS
Abstract
Nanofibrous hydrogel microarray systems that act as facile, high
throughput platforms for in vitro drug discovery and investigation
and screening of combinatorial effects of physical and biochemical
cues on maturation and differentiation of mammalian cells.
Inventors: |
Floren; Michael; (Boulder,
CO) ; Tan; Wei; (Broomfield, CO) ; Sharma;
Sadhana; (Boulder, CO) ; Bryant; Stephanie;
(Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of Colorado, a body
corporate |
Denver |
CO |
US |
|
|
Family ID: |
56367377 |
Appl. No.: |
14/991889 |
Filed: |
January 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62101334 |
Jan 8, 2015 |
|
|
|
Current U.S.
Class: |
506/9 ; 427/240;
427/430.1; 435/182; 435/397; 506/10; 506/15; 506/18; 506/19;
506/20; 506/26 |
Current CPC
Class: |
C12N 11/04 20130101;
G01N 33/5026 20130101; B01L 2300/069 20130101; B01L 2300/089
20130101; C40B 60/12 20130101; B01L 2300/123 20130101; B01J
2219/00644 20130101; B01J 2219/00743 20130101; G01N 33/5023
20130101; B01L 3/5085 20130101; C12M 25/14 20130101; B01J
2219/00385 20130101; G01N 33/502 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; B05D 1/00 20060101 B05D001/00; B05D 1/18 20060101
B05D001/18 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with Government support under grant
numbers K25HL097246 and R01 HL119371 awarded by the National
Institutes of Health (NIH). The U.S. Government has certain rights
in this invention.
Claims
1. An in vitro cell culture matrix comprising a poly(ethylene
glycol) hydrogel fiber anchored to a solid support.
2. The cell culture matrix of claim 1, wherein the hydrogel
comprises at least one of a poly(ethylene glycol) dimethacrylate
hydrogel, and a thiol-ene poly(ethylene glycol) hydrogel.
3. The cell culture matrix of claim 1, having an average fiber
diameter between about 0.1 .mu.m to about 3 .mu.m.
4. The cell culture matrix of claim 1, wherein the matrix has an
elastic modulus between about 0.4 kPa and about 15 kPa.
5. The cell culture matrix of claim 1, wherein the solid support is
glass.
6. The cell culture matrix of claim 5, wherein at least one surface
of the glass support is modified with at least one of
3-(Trimethoxysilyl)propyl methacrylate (TMPMA) and
3-(mercaptopropyl) triethoxysilane.
7. The cell culture matrix of claim 1, further comprising at least
one spot of biomaterial deposited on the electrospun hydrogel
fiber.
8. The cell culture matrix of claim 7, wherein the spot is a
microdot having a diameter between about 100 .mu.m and 500
.mu.m.
9. The cell culture matrix of claim 8, wherein the at least one
microdot is at least two microdots having a pitch to pitch distance
between about 250 .mu.m and about 750 .mu.m.
10. The cell culture matrix of claim 7, wherein the biomaterial is
at least one compound selected from a polysaccharide, a
proteoglycan, a glycosaminoglycan, a cell membrane bound protein, a
growth factor, a peptide signaling motif, a hormone, collagen I,
collagen type II, collagen III, collagen IV, fibronectin, laminin,
chitosan, elastin, entactin, fibronectin, tenascin, heparin
sulfate, chondroitan sulfate, dermaten sulfate, and karatan
sulfate.
11. The cell culture matrix of claim 1, further comprising at least
one mammalian cell.
12. The cell culture matrix of claim 9, wherein the at least one
mammalian cell is a stem cell.
13. A method of making an in vitro cell culture matrix comprising:
forming a hydrogel fiber into a 3-dimensional nanofibrous matrix;
and, anchoring the 3-dimensional nanofibrous matrix to a solid
support to form an in vitro cell culture matrix.
14. The method of claim 13, further comprising modifying the in
vitro cell culture matrix by at least one of: depositing an array
of biomaterial microdots onto the 3-dimensional nanofibrous matrix;
and, seeding a suspension of mammalian cells onto the 3-dimensional
nanofibrous matrix.
15. A method of high throughput combinatorial screening of
engineered microenvironments comprising: a. forming an in vitro
cell culture matrix comprising: i. a hydrogel fiber anchored to a
solid support; ii. biomaterial microdots arrayed on the hydrogel
fiber; and, iii. at least one mammalian cell seeded within the
hydrogel fiber, b. measuring an activity of the at least one
mammalian cell selected from gene expression, cell function,
metabolic activity, cellular morphology, and a combination
thereof.
16. The method of claim 15, wherein the forming of the in vitro
cell culture matrix comprises forming the hydrogel fiber by a
fabrication technique selected from electrospinning,
electrospraying, spin-coating, and deposition by dipping.
17. The method of claim 15, wherein the biomaterial is at least one
compound selected from a polysaccharide, a proteoglycan, a
glycosaminoglycan, a cell membrane bound protein, a growth factor,
a peptide signaling motif, a hormone, collagen I, collagen type II,
collagen III, collagen IV, fibronectin, laminin, chitosan, elastin,
entactin, fibronectin, tenascin, heparin sulfate, chondroitan
sulfate, dermaten sulfate, and karatan sulfate.
18. The method of claim 15, wherein the at least one mammalian cell
is a stem cell.
19. The method of claim 15, further comprising contacting the at
least one mammalian cell with a test agent prior to measuring an
activity of the at least one mammalian cell.
20. The method of claim 19, wherein the test agent is at least one
of a growth factor, a hormone, a putative anti-cancer compound, a
cell-surface protein inhibitor, a putative angiogenesis inhibitor,
a modulator of Epithelial-Mesenchymal Transition (EMT).
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Patent Application Ser. No.
62/101,334, filed Jan. 8, 2015 which is incorporated herein in by
reference.
TECHNICAL FIELD
[0003] The invention relates to the production of hydrogel
microarrays useful for high throughput screening of engineered
microenvironments.
BACKGROUND
[0004] There is abundant evidence that local signals from
tissue-specific extracellular matrix microenvironments
significantly affect cellular differentiation, phenotypic
expression and maintenance. Substrate biophysical signals, such as
soluble factors, cell-ligand interactions, matrix elasticity and
geometry play a critical role in a diversity of biological events
including cell adhesion, growth, differentiation, and apoptosis.
Together, these signals converge to provide a multifaceted, complex
mechano-chemical signaling environment for highly-specific tissue
morphogenesis and regeneration. Despite accumulated knowledge
regarding individual and combined roles of various mechano-chemical
ECM signals in stem cell activities, the intricacy exhibited by
cellular microenvironments poses a considerable challenge in
resolving the mechanisms ascribed to stem cell behavior and fate
processes. This complexity mandates a systemic approach whereby
integrative studies must be expanded to capture a more
comprehensive understanding of the determinants which direct stem
cell differentiation toward desired cell type and function.
Conventional methods to elucidate these mechanisms have
traditionally been executed in large scale, two-dimensional tissue
culture platforms which are often limited by combinatorial brevity,
substrate production, and reagent supply. Furthermore, these
signals, matrix and biophysical microenvironment, are often
observed independently to differentiate cells on 2D substrates, an
environment vastly different from the way cells are presented
naturally in vivo, i.e. in 3D tissue, which elicits multiple signal
inputs to regulate cell fate.
[0005] High through-put approaches have emerged in recent years to
circumvent the limitations of traditional low-through-put
techniques (i.e. conventional cultureware), with the promise of
developing complex platforms for combined biomolecule/substrate
discovery. The salient features of microarray technology include
the reproducibility and screening of multiple microenvironments
with significantly less reagent and substrate requirements than
traditional methods, while lending improved deconstruction of
complex multivariable studies. Several reports have demonstrated
ECM protein microarrays, soluble factor screening, biomaterial
chemistry screening, and multiple signal integration arrays (i.e.
elasticity and chemical factor) with encouraging results. However,
despite the versatility afforded by current microarray
technologies, the incorporation of multiple signals within
engineered microarrays remain limited, and combinatorial microarray
technologies in three-dimensions, coupled with other biophysical
properties, such as tunable stiffness and geometry, have not been
demonstrated. Capturing complex, multifaceted 3-dimensional
environments in high-throughput with combinatorial signaling will
likely prove a necessity in designing and using tissue regeneration
biomaterial platforms.
SUMMARY
[0006] This disclosure provides a microarray platform based on
electrospun nanofibrous hydrogels that can be used for screening of
microenvironmental factors that influence cell maturation and
differentiation in a high throughput manner. The nanofibrous
hydrogels may be photoclickable thiol-ene poly(ethylene glycol)
hydrogels, which polymerize by an orthogonal, step-growth mechanism
wherein one thiol reacts with one ene' leading to a highly
homogenous distribution in crosslinks. The nanofibrous hydrogels
may also be poly(ethylene glycol) dimethacrylate polymers that
undergo chain growth polymerization. The electrospun nanofibrous
hydrogel microarray platforms of this disclosure provide good
control over substrate elasticity and enable post-functionalization
of the already prepared electrospun hydrogel substrates with
extracellular matrix (ECM) molecules, such as peptides, with high
reactivity and specificity.
[0007] This disclosure provides a thiol-ene poly(ethylene glycol)
fiber cell culture substrate. The hydrogel fiber substrate may
comprise a plurality of microspot or microdot islands that comprise
one or more biomaterials. The biomaterials may include a
polysaccharide, a proteoglycan, a glycosaminoglycan, a cell
membrane bound protein, a growth factor, a peptide signaling motif,
a hormone, or combinations of these materials. The hydrogel fiber
substrate may be anchored to a solid support, such as a glass
substrate. The hydrogel fiber substrate may be seeded with
mammalian cells, such as stem cells.
[0008] This disclosure also provides methods of making a thiol-ene
poly(ethylene glycol) fiber cell culture substrate. The method
comprises forming a hydrogel fiber into a 3-dimensional nanofibrous
matrix; and, anchoring the 3-dimensional nanofibrous matrix to a
solid support to form a cell culture matrix. An array of
biomaterial microdots may then be deposited onto the 3-dimensional
nanofibrous matrix. Additionally or alternatively, a suspension of
mammalian cells may be seeded onto the 3-dimensional nanofibrous
matrix.
[0009] The culture substrates of this disclosure are useful for
culturing one or more cell types that adhere to each location
comprising an insoluble and/or soluble material (e.g., an adherence
material).
[0010] This disclosure also provides methods of high throughput
screening of engineered microenvironments by first forming an in
vitro a thiol-ene poly(ethylene glycol) hydrogel fiber cell culture
substrate anchored to a solid support, including biomaterial
microdots arrayed on the hydrogel fiber and at least one mammalian
cell seeded within the hydrogel fiber. An activity of the mammalian
cell(s) is then monitored. The cellular activity may include gene
expression, cell function, metabolic activity, cellular morphology,
or combinations thereof. These methods may include contacting the
mammalian cell(s) with a test agent prior to, or after, measuring a
cellular activity. The test agent may include a growth factor, a
hormone, a putative anti-cancer compound, a cell-surface protein
inhibitor, a putative angiogenesis inhibitor, a modulator of
Epithelial-Mesenchymal Transition (EMT), or combinations
thereof.
[0011] Other aspects of this disclosure will be understood from the
drawings, the detailed description, and examples provided
below.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1A shows rat mesenchymal stem cells (rMSCs) cultured
for 24 hours on neo-tissue protein arrays. The image is a
magnification of one printed subarray exhibiting discrete cellular
"islands." FIG. 1B shows the three-dimensional rendering of a rMSC
cellular dot formed on a cellular matrix of this disclsoure.
[0013] FIG. 2 shows the selective adhesion of rMSCs on ECM
microarrays with different elasticity, 15 min. UV exposure (top
photo) and 5 min. UV exposure (bottom photo).
[0014] FIG. 3A shows the chemical structure of 4-arm
PEG-5K-norbornene (PEGNB). FIG. 3B shows a scanning electron
microscope (SEM) image of dry and hydrated thiol-ene poly(ethylene
glycol) electrospun hydrogels of this disclosure.
DESCRIPTION OF EMBODIMENTS
[0015] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a spot" includes a plurality of such spots and reference to "the
cell" includes reference to one or more cells, and so forth.
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and
materials are described herein.
[0017] The publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application.
[0018] The need for engineered stem cell niches integrating several
extrinsic stimuli has become a significant challenge within the
research community. Due to the lack of current traditional methods
to accurately and efficiently capture these complex
microenvironments we adopted a high throughput method whereby
matrix physical properties and biological ligands could be
modulated. The design of a multivariate protein microarray for
screening of SC microenvironments required the fabrication of an
appropriate platform incorporating a three-dimensional substrate
with nanofiber architecture and tunable elasticity and finally the
integration of a combinatorial ECM protein microarray upon our
engineered substrates.
[0019] The cellular microenvironment plays a critical role in
determining cell fate and function. Extracellular determinants of
survival, proliferation, migration, and differentiation include
soluble signals (cytokines, dissolved gasses), insoluble cues
(extracellular matrix, cell-cell interactions, biomaterials), and
physical stimuli (shear stress). Miniaturization of bioassays using
multiwell plates and robotic liquid handling enables combinatorial
screening of the effects of soluble species on cellular behavior;
however, analogous approaches for screening the effects of
insoluble cues are in their infancy. Cellular interactions with the
extracellular matrix (ECM) are of particular interest as ligation
of an integrin can directly induce cellular signaling, modulate the
response to other agonists, and influence the behavior of other
integrins, a phenomenon called crosstalk. Thus, the extracellular
matrix plays a role in developing an integrated picture of the
microenvironment in the fate of many diverse cell types.
[0020] Cell-ECM interactions have been studied using several
approaches. Typically, purified matrix proteins are adsorbed to
cell culture substrates alone or in a combination requiring on the
order of 10.mug of protein per 96-well plate. These `2-dimensional`
approaches are complemented by so-called `3-dimensional` approaches
such as embedding cells within ECM gels. More complex ECM has also
been investigated using cell-derived matrix in vitro or
decellularized tissue sections. In addition to natural ECM
components, biomaterial approaches have yielded several hybrid
matrices with tethered biomolecules and tunable degradation in a
3-dimensional hydrogel context. Studies of the interaction of
cell-ECM provides a critical first step towards developing a
comprehensive understanding of insoluble cues in the cellular
microenvironment.
[0021] Growth factor signals synergistically interact in permissive
ECM microenvironments. Cross talk between ECM proteins and soluble
growth factors would be best investigated using a highly parallel
microfluidic platform integrating robotic spotting of substrates on
a 3-dimensional cellular matrix, generating combinatorial soluble
factor mixtures. Such a platform can be used in other experiments
to investigate other cellular pathways involving multiple soluble
factor interactions and integrin cross talk to study the combined
effect of soluble and insoluble factors on cell fate and
function.
[0022] This disclosure provides a robust method to create
3-dimensional cell matrices composed of hydrogel fibers that are
spotted with protein or other biomaterials in an array using a
spotter device (e.g., a DNA spotter device). The design of this
microarray substrate was an important requisite of our studies of
the effects of ECM, which necessitated a cellular matrix with
tunable elasticity, three-dimensional architecture, reproducible
fabrication, and ease of sample production. The cell culture
matrices of this disclosure provide each of these advantages as
well as other advantages described below.
[0023] Culturing mammalian cells, including especially stem cells,
on combinatorial mixtures of extracellular matrix (ECM) proteins in
the methods and cell matrices of this disclosure yields novel
insights into the role of the microenvironment that may not be
available using conventional 2-dimensional tissue culture methods.
The methods and systems of this disclosure are amenable to
depositing almost any insoluble or soluble material/biological
material, such as polysaccharides, proteoglycans,
glycosaminoglycans, membrane bound proteins, DNA, siRNA, and
tethered growth factors or peptide signaling motifs. The methods
and systems can also be easily adapted to: exploit lineage-specific
fluorescent reporter strategies, co-cultivation of epithelia and
stroma, and/or combinations of soluble factors to screen the
effects of growth factors or small molecules in conjunction with
underlying ECM structure and protein chemistry.
[0024] The cell culture matrix of this disclosure comprises a
biologically compatible fiber anchored to a substrate or solid
support. The fiber may be natural material (such as collagen,
gelatin, elastin), or a synthetic polymer (such as PCL, PLGA, PLA,
PS, PES), and/or synthetic and natural hydrogels (such as PEG-based
hydrogels, silk protein). Hydrogels provide for hydration of bound
cells, lack of diffusion of insoluble materials, low background
binding of cells and free flow of cells across the surface of the
microarray due to weak cell repulsion. The hydrogel fiber may be a
poly(ethylene glycol) (PEG) polymer fiber. The hydrogel fiber may
include a poly(ethylene glycol) dimethacrylate hydrogel, and/or a
thiol-ene poly(ethylene glycol) hydrogel. Other examples of
hydrogels useful in the methods and systems of this disclosure may
include polyvinylalcohol (PVA), physically cross-linked by partial
crystallization of the chain and/or hydrogels based on segmented
polyurethanes or polyureas, polypeptide or polysaccharide
hydrogels, such as agarose or cross-linked hyaluronic acid,
partially hydrolyzed or aminolyzed polyacrylonitrile (PAN), so long
as the hydrogel materials are sufficiently biocompatible and do not
release harmful substances.
[0025] The hydrogel fiber may have an average fiber diameter
between about 0.1 .mu.m to about 3 .mu.m. The hydrogel fiber may
have an average fiber diameter between about 0.2 .mu.m to about 0.6
.mu.m in the dry state. The hydrogel fiber may have an average
fiber diameter between about 0.4 .mu.m to about 3 .mu.m in the wet,
or hydrated state. The hydrogel fiber may have an elastic modulus
between about 0.4 kPa to about 15 kPa. The hydrogel fiber may have
an elastic modulus between about 1 kPa to about 5 kPa.
[0026] The substrate used in the methods and systems of this
disclosure can be made of any material suitable for culturing
mammalian cells. For example, the substrate can be a material that
can be easily sterilized, such as plastic or other artificial
polymer material, so long as the material is biocompatible. The
substrate can be any material that allows the hydrogel fibers to
adhere (or can be modified to allow the hydrogel fibers to adhere,
or not adhere at select locations). Various substrates or solid
supports can be used to anchor the hydrogel fiber in the methods
and systems of this disclosure. Such substrates include, but are
not limited to, glass, plastics such as polystyrene and/or
polypropylene, metals such as stainless steel, silicon and the
like, including, but not limited to, polyamides; polyesters;
polystyrene; polypropylene; polyacrylates; polyvinyl compounds
(e.g. polyvinylchloride); polycarbonate (PVC);
polytetrafluoroethylene (PTFE); nitrocellulose; cotton;
polyglycolic acid (PGA); cellulose; dextran; gelatin, glass,
fluoropolymers, fluorinated ethylene propylene, polyvinylidene,
polydimethylsiloxane, polystyrene, and silicon substrates (such as
fused silica, polysilicon, or single silicon crystals), and the
like. Also metals, such as gold, silver, or titanium films, can be
used.
[0027] The choice of the solid support should be taken in to
account where biocompatibility with the cells or biomaterials or
drugs that will be used in the cellular matrices may be effected.
The solid support can be chosen from any number of rigid or elastic
supports. For example, the solid support can comprise glass or
polymer microscope slides.
[0028] At least one surface of the solid support may be treated or
modified with a chemical, such as 3-(Trimethoxysilyl)propyl
methacrylate (TMPMA), and/or a silicone compound such as
3-(mercaptopropyl) triethoxysilane. The solid support may also be
modified with other chemicals, such as other acrylamide compounds,
agarose, pluronics, serum albumin, or polyethylene glycol. The
substrate may be densely hydroxylated prior to silanization, a
highly porous glass substrate, or alternate silane coupling
agents.
[0029] Alternatively, the hydrogel can be modified such that cell
attachment is inhibited for a short period of time or in specific
regions. During this period or in these regions, a spotter device
may be used to deposit an etching solution (such as a mild periodic
acid solution) to a hydrogel surface containing a degradable
component. These regions can then be further modified by
subsequently depositing protein solutions or pre-polymer solutions
(with or without proteins) to the locations. Proteins or
pre-polymer solutions can thus be immobilized on the solid support
using photo-gelation or chemical crosslinking. Additionally,
degradable polymer matrices can be incorporated into the hydrogel
substrate that would allow for local sustained release of soluble
factors. Each region may be tailored individually using these
techniques.
[0030] The hydrogel fibers may be tuned or formed to retain
specific biological molecules including, but not limited to,
proteins, peptides, oligonucleotides, polynucleotides,
polysaccharides, lipids and other biological molecules (i.e.,
"biomaterials"). A deformable hydrogel can be used in the methods
and systems of this disclosure. Deformable hydrogels include
polyacrylamide hydrogels. The hydrogel may include components that
weakly repulse cells, thereby providing low background binding. The
substrate and/or the hydrogel fibers may comprise a polymerized
mixture including acrylamide and hydrophilic acrylates.
[0031] Hydrogels may be selected such that specific binding of
desired biomaterials, including specific cell types is promoted and
non-specific binding is reduced. Those of skill in the art will
understand that cells vary in their ability to adhere to a cell
culture matrix material and/or substrate material.
[0032] The hydrogel fibers acts as a deformable membrane that
allows for seeded cells to be actively stretched during culture.
Membrane deformation can be controlled using any of a multitude of
suitable methods. These include MEMS motors incorporated into the
cell culture matrix or on the solid support, and connected to the
culture substrate and electroactive polymers that respond to
electric field by undergoing a shape change. Examples of such
materials include electrostrictive materials such as thin acrylate
films with deformable electrodes placed on both sides of the
material. An applied electric field causes the acrylate film to
compress or expand, resulting in a concurrent change in surface
area such that the total volume of the film remains constant. Such
materials are known as "electrostrictive" in the field of
electroactive polymers. Methods for generating force and
deformation in a pliable material using electric fields
("electrophoretic"), or alternating non-uniform electric fields
("dielectrophoretic") are also possible by using a material that is
responsive to such modes of excitation. As an example, the hydrogel
fiber may incorporate charged particles, or neutral particles that
can experience an induced dipole force in the presence of a uniform
or non-uniform electric field. The electric field can be generated
by placing the gel material on an electrode array, which can apply
a static electric field, or an alternating electric field of the
appropriate frequency to induce a net force on the dielectric
medium of the gel.
[0033] By deformable is meant that a deformable material is capable
of being damaged by contact with a rigid instrument. Examples of
deformable materials include hydrogels, polyacrylamide, nylon,
nitrocellulose, polypropylene, polyester films, such as
polyethylene terephthalate, and the like. Non-deformable materials
include materials that do not readily bend, and include glass,
fused silica, nanowires, quartz, plastics (e.g.
polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate,
and blends thereof) and the like; metals (e.g. gold, platinum,
silver, and the like). A deformable material may be layered upon a
non-deformable material.
[0034] The hydrogel fiber may be produced as a fiber on a solid
substrate (e.g., a glass slide, cell culture plate, and the like),
by mixing a solution of a monomer, a crosslinker, and a catalyst
and/or an initiator and forming a fiber. The monomer and
crosslinker may be prepared in one solution of an amount of
initiator added. The solution is polymerized and crosslinked either
by ultraviolet (UV) radiation or other appropriate UV conditions,
or by thermal initiation at elevated temperature. The elasticity of
the polymeric fiber matrix is controlled, or "tuned", by changing
the amount of crosslinker and/or the percent solids in the monomer
solution, and/or the exposure to UV/light. The polymerization may
also be initiated by UV/light exposure.
[0035] The fiber may be formed by a fabrication technique such as
electrospinning, electrospraying, spin-coating, and/or deposition
by dipping.
[0036] Following fabrication, unpolymerized monomer is washed away,
typically with water, leaving a nanofibrous gel matrix. Further
lithographic techniques known in the semiconductor industry can be
used to generate patterned structures in the nanofibrous matrix.
Light may be applied to discrete locations on the matrix to further
tune or activate specified regions, for example for the attachment
of an oligonucleotide, an antibody, an antigen, a hormone, hormone
receptor, a ligand or a polysaccharide on the matrix.
[0037] For hydrogel-based arrays using polyacrylamide, biomolecules
can be prepared by forming an amide, ester or disulfide bond
between the biomolecule and a derivatized polymer comprising the
cognate chemical group. Covalent attachment of the biomolecule to
the polymer is usually performed after polymerization and chemical
cross-linking of the polymer is completed.
[0038] Controlled-release of soluble factors from a degradable
polymer substrate has been demonstrated in the fields of drug
delivery and biomimetic engineered surfaces. Typically, soluble
factors are immobilized within a polymer matrix or hydrogel. As the
matrix degrades, soluble factors are released into the environment.
The degradation of the polymer, and thus the release kinetics, can
be tailored by modifying the composition of the polymer or
hydrogel. In one variation, growth factors are incorporated into
poly(lactide-co-glycolide) (PLGA) microspheres. The GF-laden
microspheres are then incorporated into a suitable matrix, such as
PEG-hydrogel, PLGA, or acrylamide.
[0039] The nanofibrous matrix may be modified to promote cellular
adhesion and growth. For example, the fiber may be treated with
protein (i.e., a peptide of at least two amino acids) such as
collagen or fibronectin to assist cells in adhering to the
substrate. The proteinaceous material may be used to produce an
array in or on the substrate. The array produced by the protein
serves as a "template" for formation of a cellular microarray. A
single protein may be adhered to the fiber, although two or more
proteins may be used to spot the fiber using a device, such as a
spotter device. Proteins that are suitable for use in modifying the
fiber to facilitate cell adhesion include proteins to which
specific cell types adhere under cell culture conditions, for
example, collagen, fibronectin, gelatin, collagen type IV, laminin,
entactin, and other basement proteins, including glycosaminoglycans
such as heparin sulfate. Combinations of such proteins may also be
used.
[0040] Depositing or patterning biomaterials on the nanofibrous
matrix may be achieved using various micro-spotting techniques.
Spotting techniques involve the precise placement of materials at
specific sites or regions using automated techniques. Conventional
physical spotting techniques such as quills, pins, or
micropipettors are able to deposit material on the matrix in the
range of 10 to 250 microns in diameter (e.g., about 100
spots/microwells per well of a 96 well culture plate). In some
instances, the density can be from 400 to 10000 spots per square
centimeter, allowing for clearance between spots. Lithographic
techniques, such as those provided by Affymetrix (e.g., U.S. Pat.
No. 5,744,305, the disclosure of which is incorporated by reference
herein) can produce spots down to about 10 microns square, with no
clearance between spots, resulting in approximately 800,000 spots
per square centimeter. Insoluble and/or soluble materials may be
spotted in very small, e.g. nanoliter, increments using a spotting
device. The spotting device may employ one or more piezoelectric
pumps, acoustic dispersion, liquid printers, micropiezo dispensers,
or the like to deliver such reagents/biomaterials. The spotting
device may comprise an apparatus and method like or similar to that
described in U.S. Pat. Nos. 6,296,702, 6,440,217, 6,579,367, and
6,849,127. An automated spotting device may be used (e.g. Perkin
Elmer BIOCHIP ARRAYER.TM.. A number of contact and non-contact
microarray printers are available and may be used to dispense/print
the soluble and/or insoluble biomaterials on the nanofibrous
matrix. For example, BIOCHIP ARRAYER.TM., Labcyte and IMTEK
TOPSPOT.TM., and Bioforce.TM.. These devices utilize various
approaches to non-contact spotting, including piezo electric
dispension; touchless acoustic transfer; en bloc printing from
multiple microchannels; and the like. Other approaches include ink
jet-based printing and microfluidic platforms. Contact printers are
commercially available from TeleChem International (ARRAYLT.TM.).
Non-contact printers are of particular interest because they are
more compatible with deformable hydrogel surfaces and allow for
simpler control over spot size via multiple dispensing onto the
same location.
[0041] Non-contact printing will typically be used for the
production of arrays of dots (microdots) on the nanofibrous matrix.
By utilizing a printer/spotter that does not physically contact the
surface of substrate, no aberrations or deformities are introduced
onto the surface, thereby preventing uneven or aberrant cellular
capture at the site of the spotted material. Such printing methods
find particular use with hydrogel substrates. Printing methods of
interest, including those utilizing acoustic or other touchless
transfer, also provide benefits of avoiding clogging of the printer
aperature, e.g. where probe solutions have high viscosity,
concentration and/or tackiness. Touchless transfer printing also
relieves the deadspace inherent to many systems. The use of print
heads with multiple ports and the capacity for flexible adjustment
of spot size can be used for high-throughput, automated microarray
preparation.
[0042] The total number of spots on the matrix will vary depending
on the number of different conditions (e.g., material combinations)
to be explored, as well as the number of control spots, calibrating
spots and the like, as may be desired. Generally, the pattern
present on the surface of the matrix will comprise at least 2
distinct spots, usually about 10 distinct spots, and more usually
about 100 distinct spots, where the number of spots can be as high
as 50,000 or higher. The spot/microdot will usually have an overall
circular dimension and the diameter will range from about 10 .mu.m
to 5000 .mu.m, or from about 100 .mu.m and 500 p.m.
[0043] Following the formation of the array of microdots of
biomaterial(s), the nanofiberous cell culture matrices may be
subjected to a second exposure to UV light to covalently cross link
the deposited/arrayed biomaterials to the fiberous matrix. This is
particularly useful for covalently linking sulfhydryl groups of
proteins or peptides to the fiberous matrix. In this way, other
molecules of interest, such as dyes or florescent molecules, can be
covalently linked to the fiberous matrix by first attaching the
molecule to a peptide linker that will cross link the molecule to
the fiberous matrix following UV exposure. Additionally or
alternatively, molecules may also be grafted to the fiberous matrix
after being functionalized. For example, a biomolecule may be
treated with oxygen or ammonia plasma to functionalize the
molecule, which is then grafted to the fiberous matrix.
[0044] The methods and systems of this disclosure are useful to
modulate the density of biological materials "spotted" on a cell
culture substrate. For example, the maximum density of ECM
molecules is dependent on several factors including: the
concentration of stock solution, the solubility of ECM proteins,
the porosity of the fiber matrix, and the mode of deposition (e.g.,
pin or piezoelectric). Controlling the amount/density of biological
materials in a culture environment can modulate cell growth and
differentiation. Accordingly, the spotting device can be calibrated
and used to provide specific amounts of insoluble and/or soluble
biological materials to select "spots" or microdots.
[0045] This disclosure provides methods and systems useful for
identifying optimal conditions for controlling cellular development
and maturation. For example, the methods and systems of this
disclosure are useful for identifying optimal conditions that
control the fate of cells (e.g., differentiating stem cells into
more mature cells, maintenance of self-renewal, and the like) by
controlling and optimizing the extracellular and soluble
microenvironment upon which the cells are cultured in parallel
array fashion for rapid high throughput techniques. A 3-dimensional
cell culture microarray platform of this disclosure is useful for
testing a multitude of soluble factors (e.g., growth factors,
hormones, steroids and the like) and insoluble factors (e.g.,
extracellular matrix, cell adhesion proteins, glycoproteins and the
like) individually and in combination using minimal reagents and a
relatively small numbers of cells, in an automated and high
throughput methodology.
[0046] With the advent of DNA robotic spotting technology, it is
now possible to routinely deliver nanoliter volumes of many
different materials, from interfering RNA, to peptides, to
biomaterials at precise locations on a microarray substrate. ECM
materials may be more difficult to manipulate due to, for example,
incompatible process conditions for ECM protein spotting, extensive
customization of spotting equipment, or lack of pattern fidelity
(i.e. cell localization) over time. These limitations may be
overcome by, for example, modifying the buffer used in a spotting
device to allow for accurate ECM deposition, and the use of
nanofiberous materials that permit ECM immobilization, such as
hydrogel materials. The methods and systems of this disclosure are
useful for spotting substantially purified or mixtures of
biological proteins, nucleic acids and the like (e.g., collagen I,
collagen III, collagen IV, laminin, and fibronectin) in various
combinations on a standard cell culture substrate (e.g., a
microscope slide) using commercially available chemicals and a
conventional DNA robotic spotter. Thus, the systems and methods of
this disclosure allow for a multitude of insoluble factors (e.g.
extracellular matrix, biomaterials), tethered soluble factors
(e.g., growth factors), or mixtures of insoluble and soluble cues
to be tested in parallel on a small scale.
[0047] This disclosure provides a nanofiberous matrix comprising
multiple distinct cell culture domains of arbitrary protein or
polymer composition and size by using robotic spotting technology
(e.g., a DNA spotting device or a similar device), to transfer
nanoliter quantities of biomaterials onto a fiber matrix anchored
to a solid substrate (e.g., glass, silicon, polymer or other
biocompatible material used in cell culture) at desired
locations.
[0048] One or more desired biological materials may be deposited as
discrete "spots" on the fiberous matrix. Each spot may comprise a
different biological material composition. Each spot may comprise
the same biological material composition. Cells cultured on the
spots may be the same or different. For example, a defined ECM
material is deposited as discrete spots onto a cell culture matrix
of this disclosure, and cells are then seeded within the matrix and
cultured under desired culture conditions. Where the spots comprise
different biological materials, the cells experience different
stimuli while being cultured simultaneously but maintained in
distinct spatial domains creating a cellular array.
[0049] The biomaterials or test analytes delivered to the cells in
the cellular microarrays formed in the methods and systems of this
disclosure may include organic and inorganic molecules, including
biological molecules. For example, the analyte may be an
environmental pollutant (including pesticides, insecticides,
toxins, and the like); a chemical (including solvents, polymers,
organic materials, and the like); therapeutic molecules (including
therapeutic and abused drugs, antibiotics, and the like);
biological molecules (including, e.g., hormones, cytokines,
proteins, lipids, carbohydrates, cellular membrane antigens and
receptors (neural, hormonal, nutrient, and cell surface receptors)
or their ligands); whole cells (including prokaryotic and
eukaryotic cells; viruses (including, e.g., retroviruses,
herpesviruses, adenoviruses, lentiviruses); spores; and the
like.
[0050] One cell type or a plurality of cell types can be used in
the microarrays formed by the methods and systems of this
disclosure. For example, cultures of two cell types can be
performed thus allowing for co-culture of two cell types. For
example, the fiberous matrix surrounding a first spot can be
modified such that cells cannot attach to, or migrate to, the
regions surrounding the spot. In this manner, mono-culture or
co-culture involving multiple cell types and multiple ECM coatings
can be tested in a single nanofiberous matrix system.
[0051] The type of biological materials (e.g., ECM materials)
deposited in the microarray will be determined, in part, by the
cell type or types to be cultured. For example, ECM molecules found
in the hepatic microenvironment are useful in culturing
hepatocytes, the use of primary cells, and a fetal liver-specific
reporter ES cell line. It will be recognized that the biomaterial
used in the array may be modulated by cellular interactions. For
example, interaction with ECM is known to modulate matrix
metalloproteinase expression, integrin activity, and matrix
expression.
[0052] The effect of soluble and insoluble factors/test analytes
and/or biomaterials on cells cultured in a microarray formed by the
methods and systems of this disclosure can be probed or evaluated
using a specific marker, or examined for cellular morphology, and
the like. For example, the array can be probed for the state of
differentiation using various techniques including in situ
hybridization, antigenic recognition (intracellular or cell
membrane), in situ PCR, or an artificial DNA-reporter construct
such as GFP or beta-galactosidase. The cell array can be assessed
using fluorescent microscopy, high resolution light microscopy, a
confocal laser scanner (such as those commonly used for DNA
microarray applications), fluorescence and absorbance plate
readers, scanners, or other such equipment. In many aspects, the
measurements will be made by automated microscopes, plate readers
and the like. In this manner, "optimal" culture conditions, as
defined by the user, can be identified for closer examination and
testing using more conventional techniques.
[0053] Cells cultured in microarrays of the disclosure may be used
to study cell and tissue morphology. For example, enzymatic and/or
metabolic activity may be monitored in the culture by fluorescence
or spectroscopic measurements on a conventional microscope. In one
aspect, a fluorescent metabolite in the fluid/media is used such
that cells will fluoresce under appropriate conditions (e.g., upon
production of certain enzymes that act upon the metabolite, and the
like). Alternatively, recombinant cells can be used in the cultures
system, whereby such cells have been genetically modified to
include a promoter or polypeptide that produces a therapeutic or
diagnostic product under appropriate conditions (e.g., upon
zonation or under a particular oxygen concentration).
[0054] Embryonic stem cells are a potential source of
differentiated cells that could be used in cell therapy, drug
discovery, and basic research. Current methods for differentiating
embryonic stem cells in vitro are generally inefficient for
generating specific lineages, and rely on the use of heterogeneous
cell aggregates called embryoid bodies. Exceptions to this
generalization are a few rare reports of efficient monolayer
culture methods, underscoring the importance of a tightly regulated
environment for efficient lineage-specific differentiation. While
most studies focus on growth factors, the importance of ECM in
developmental processes has increasingly been recognized. In vitro,
undifferentiated mouse ES cells express integrins .alpha.6,
.beta.1, .beta.4, .beta.5, laminin receptor 1, and dystroglycan;
signals from ECM. Stem cell differentiation studies could therefore
benefit from a parallelized culture platform of this disclosure.
Monitoring can be performed using specific markers or ubiquitous
cellular constituents such as actin and keratin.
[0055] This disclosure provides a cell culture matrix that can be
read or analyzed by a variety of methods including, but not limited
to, surface plasmon resonance, mass spectrometry, quartz crystal
resonance, electron microscopy and scanning probe microscopy. In
one aspect, scanning probe microscopy (SPM) such as atomic force
microscopy (AFM). Use of an AFM or another type of SPM creates a
methodology for a simple rapid, sensitive and high throughput
method for detection of microorganisms, pathogens, biological
matter, viruses, or microparticles. This method can be used to
detect changes in a spot sample. Additionally, fluorescence or
other methods commonly practiced for detection of biological events
can be employed in the methods and systems of this disclosure.
[0056] This disclosure provides cell culture technology that can be
useful for a variety of purposes, such as determining the
appropriate culture conditions for differentiating stem cells into
more mature cells, studying cell-matrix and growth factor
interactions in a systematic manner, and potentially screening new
drug molecule candidates for their effects on cells in vitro by
immobilizing small volumes in degradable matrices for sustained
release. Additionally, the platform can be extended for use with
non-stem cells, such as primary cells (e.g. hepatocytes,
fibroblasts), genetically modified cells, and transformed or
cancerous cell types. A number of uses of the methods and systems
will be readily apparent to one of skill in the art. For example,
stem cell therapeutic companies could use such technology to
optimize differentiation protocols for specific lineages.
Lifescience or pharmaceutical companies could use such technology
for optimizing in vitro production of recombinant proteins.
Pharmaceutical companies could use a miniaturized cell culture
platform to test toxicity of potential drug compounds in a parallel
manner. Researchers could use such a platform to test the effects
of insoluble or tethered soluble and insoluble cues on cellular
differentiation.
[0057] The culture system and microarrays of the disclosure can be
used in a wide variety of applications. These include, but are not
limited to, screening compounds, growth/regulatory factors,
pharmaceutical compounds, and the like, in vitro; elucidating the
mechanisms of certain diseases, studying the mechanisms by which
drugs and/or growth factors operate, diagnosing and monitoring
cancer in a patient, and the production of biological products.
[0058] The methods and systems of the disclosure may be used in
vitro to screen a wide variety of compounds, such as cytotoxic
compounds, growth/regulatory factors, pharmaceutical agents, and
the like, to identify agents that modify cell (e.g., hepatocyte)
function and/or cause cytotoxicity and death or modify
proliferative activity or cell function. For example, the culture
system may be used to test adsorption, distribution, metabolism,
excretion, and toxicology (ADMET) of various agents. The activity
of a compound can be measured by its ability to damage or kill
cells in culture or by its ability to modify the function of the
cells. This may readily be assessed by vital staining techniques,
ELISA assays, immunohistochemistry, and the like. The effect of
growth/regulatory factors on the cells (e.g., hepatocytes,
endothelial cells, epithelial cells, pancreatic cells, astrocytes,
muscle cells, cancer cells) may be assessed by analyzing the
cellular content of the culture, e.g., by total cell counts, and
differential cell counts or by metabolic markers such as MTT and
XTT. This may also be accomplished using standard cytological
and/or histological techniques including the use of
immunocytochemical techniques employing antibodies that define
type-specific cellular antigens. The effect of various drugs on
normal cells cultured in the culture system may be assessed. For
example, drugs that affect cholesterol metabolism, e.g., by
lowering cholesterol production, could be tested on a liver culture
system.
[0059] The cytotoxicity to cells in culture (e.g., human
hepatocytes) of pharmaceuticals, anti-neoplastic agents,
carcinogens, food additives, and other substances may be tested by
utilizing the cell culture microarrays of this disclosure.
[0060] This disclosure also provides a screening method comprising,
generating a microarray of biomaterials on a nanofiberous cell
culture matrix of this disclosure using a spotting device (e.g., a
DNA spotting device) or similar device. A material spotted by the
device may include soluble and/or insoluble factors that have known
activity or effects on cells or may comprise factors having unknown
activity of effects on cells. Cells are then contacted with the
micro-spot array and a stable, growing culture is established. The
cells may then be examined or alternatively, the cells are exposed
to varying concentrations of a test agent. After incubation, the
culture is examined to determine the effect of the material and/or
test agent on a cell's morphology, growth, activity, and the like.
Cytotoxicity testing can be performed using a variety of supravital
dyes to assess cell viability in the liver culture system, using
techniques well-known to those skilled in the art.
[0061] Similarly, the beneficial or deleterious effects of drugs or
biologics may be assessed using the nanofiberous cell culture
matrix of this disclosure. For example, growth factors, hormones,
or drugs which are suspected of having the ability to enhance cell
or tissue function, formation or activity can be tested. In this
case, stable cultures are exposed to a test agent. After
incubation, the cultures are examined for viability, growth,
morphology, cell typing, and the like, as an indication of the
efficacy of the test substance. Varying concentrations of the drug
may be tested to derive a dose-response curve.
[0062] The culture systems of the disclosure may be used as model
systems for the study of physiologic or pathologic conditions and
to optimize the production of a specific protein.
[0063] The microarray culture system may also be used to aid in the
diagnosis and treatment of malignancies and diseases. For example,
a biopsy of a tissue (such as, for example, a cell biopsy) may be
taken from a subject suspected of having a malignancy or other
disease or disorder. The biopsy cells can then be cultured under
appropriate conditions (e.g., defined factors spotted on an array)
where the activity of the cultured cells can be assessed using
techniques known in the art. In addition, such biopsy cultures can
be used to screen agent that modify the activity in order to
identify a therapeutic regimen to treat the subject. For example,
the subject's tissue culture could be used in vitro to screen
cytotoxic and/or pharmaceutical compounds in order to identify
those that are most efficacious; i.e. those that kill the malignant
or diseased cells, yet spare the normal cells. These agents could
then be used to treat the subject.
[0064] Similarly, the beneficial effects of drugs may be assessed
using a microarray in vitro; for example, growth factors, hormones,
drugs which enhance hepatocyte formation or activity can be tested.
In this case, microarray cultures may be exposed to a test agent.
After incubation, the microarray cultures may be examined for
viability, growth, morphology, cell typing, and the like as an
indication of the efficacy of the test substance. Varying
concentrations of the drug may be tested to derive a dose-response
curve.
[0065] Using micropatterning of co-cultures and reagents can lead
to a cell or tissue model that can be optimized for specific
physiologic functions including, for example, synthetic, metabolic,
or detoxification function (depending on the function of interest)
in hepatic cell cultures.
[0066] The methods and systems of this disclosure may utilize
co-cultures of cells in which at least two types of cells are
configured in a micropattern on a substrate. Micropatterning
techniques may be used to modulate the extent of heterotypic
cell-cell contacts. In addition, co-cultures (both micropatterned
co-cultures and non-micropatterned co-cultures) have improved
stability and thereby allow chronic testing (e.g., chronic toxicity
testing as required by the Food and Drug Administration for new
compounds). Because micropatterned co-cultures are more stable than
random cultures the use of co-cultures and more particularly
micropatterned co-cultures provide a beneficial aspect to the
cultures systems of the disclosure. Furthermore, because drug-drug
interactions often occur over long periods of time the benefit of
stable co-cultures allows for analysis of such interactions and
toxicology measurements.
[0067] Typically, in practicing the methods of this disclosure, the
cells are mammalian cells, although the cells may be from two
different species (e.g., pigs, humans, rats, mice, and the like).
The cells can be primary cells, stem cells, or they may be derived
from an established cell-line. Although any cell type that adheres
to a substrate can be used in the methods and systems of the
disclosure (e.g., parenchymal and/or stromal cells), exemplary cell
include, stem cells, epithelial cells, endothelial cells, muscle
cells, neuronal cells, etc.
[0068] The methods and systems of this disclosure have been
demonstrated as set forth in more detail in the following Examples.
The working examples provided below are to illustrate, not limit,
the disclosure. Various parameters of the scientific methods
employed in these examples are described in detail below and
provide guidance for practicing the disclosure in general.
EXAMPLES
Example 1
Preparation and Characterization of a Polyethylene Glycol
Dimethacrylate (PEGDM) Cell Culture Matrix
Fabrication of PEGDM Soft Matrices
[0069] Polyethylene glycol dimethacrylate (PEGDM) with a molecular
weight of 750 kDa and polyethylene oxide (PEO) (MW 400,000 kDa)
were purchased from Sigma (Sigma-Aldrich, St. Louis, Mo.). An
electrospinning solution composed of 3.2% wt PEGDM 750, 3.4% wt
PEO, 0.4% wt of Irgacure 2959 and 93% DI H.sub.2O was mixed for 30
minutes with magnetic stir bar. PEGDM 750 photopolymerizable soft
matrices were fabricated by electrospinning on a custom set up
composed of a high voltage power supply, grounded collecting
surface, motorized syringe pump, and a 14 mm syringe. The solution
(2 ml) was spun at a distance of 26 cm from the stationary
collecting surface, at a voltage of 30 kV, and a flow rate of 1.10
ml/hr. Electronspun matrices were deposited onto glass slides 25
mm.times.75 mm (Fischer Scientific) modified with
3-(Trimethoxysilyl)propyl methacrylate (TMPMA) (Sigma) to present
methacrylate groups that bond the substrate to the glass.
Substrates were subsequently introduced into an inert argon
environment to remove oxygen followed by stabilization under UV
exposure (352 nm light) with an average intensity of 5 mW/cm.sup.2
for specific time durations.
Characterization of PEGDM Soft Matrices
[0070] FTIR Analysis:
[0071] PEGDM electrospun net samples were first loaded into a
sealed liquid-cell (Sigma) in the presence of an inert argon
environment to prevent oxygen contamination during IR acquisition.
PEGDM double bond conversion was evaluated using a real-time
mid-range fourier transform infrared spectroscopy (FTIR) (Nicolet
4700, Thermo Fisher Scientific, Waltham, Mass.) by examining the
disappearance of the C.dbd.C peak within the acrylate group
(.about.1635) on a dry specimen in the presence of UV light (15
mW/cm2) over time. To account for sample and background variation,
data were normalized with the C.dbd.O peak located in the range
from 1650 to 1726 cm.sup.-1.
[0072] Scanning Electron Microscopy Imaging:
[0073] Scanning electron microscopy (FESEM, JSM-7401F, Jeol Ltd,
Tokyo, Japan) was used to examine the microstructure of the
electrospun PEGDM substrates in both dry and hydrated states. For
hydrated samples, substrates were photopolymerized for 15 min and
rinsed in DI H.sub.2O for 24 hr. To prepare for imaging, rinsed
samples were shock froze in liquid nitrogen (-195 C) and
lyophilized for approximately 24 hr. Image) was used to analyze
changes in fiber diameter and porosity.
[0074] Fluorescent Imaging:
[0075] PEGDM soft matrices were imaged under fluorescence to
observe their properties in the wet state. Rhodamine-methacrylate
was introduced into the electrospun fibers and subsequently
stabilized during UV exposure to provide fluorescence of the
fibrous structure. PEGDM-rhodamine conjugates were then imaged
using either a fluorescent microscope or a confocal laser scanning
microscope.
[0076] Rheology:
[0077] Changes in storage modulus (G') of PEGDM substrates with
respect to photopolymerization time were characterized using a
rheometer (=5%, =1 rad/s for linear viscoelastic regime, ARES TA
rheometer, TA Instruments, New Castle, Del.). PEGDM substrates,
approximately 0.3 mm thickness) were deposited onto
3-(Trimethoxysilyl)propyl methacrylate modified circular coverslips
(D=18 mm) and photopolymerized for 2, 5, 10, or 15 min and then
rinsed in DI H.sub.2O for 24 hr. PEGDM soft matrices were tested
with a parallel plate configuration. A vertical load of 5 grams was
applied to all samples to prevent slippage. A strain sweep at a
frequency of 1 rad/s and a frequency sweep at a strain of 5% were
run on each sample. Specimens were inspected for slippage or
tearing post shearing, and data collected from the linear
visco-elastic region (LVE) in the strain sweep were used to
determine the storage modulus G'.
ECM Protein Microarray Preparation
[0078] A printing buffer consisting of 1% glycerol and 0.2% Triton
X-100 was utilized for all protein depositions. To prepare ECM
arrays, stock solutions of rat collagen I (Millipore), human
collagen III (Sigma), human collagen IV (Sigma), human fibronectin
(Millipore), mouse laminin (Sigma), and bovine elastin (Elastin
Products Company) were suspended at a concentration of 250 m/ml in
printing buffer. Factorial analysis was performed to determine 64
distinct combinations from the 6 ECM proteins of interest and
subsequently transferred to a predefined 384-well plate
configuration. Samples were deposited on the PEGDM substrate matrix
using an Aushon 2470 arrayer with 185 micron pins (Aushon
BioSystems, Billerica, Mass.), to achieve a nominal diameter of 250
microns. Individual spots with 7 replicates (8 total) of each
protein combination were deposited with a 500 .mu.m pitch distance
onto the PEGDM substrates. Between different sample depositions,
the print needles were cleaned by sonication in cleaning solution
before use. Approximately twenty ECM microarrays could be deposited
simultaneously in this method within .about.1 hr. Prepared ECM
microarrays were stored at 4.degree. C. in a humid environment for
24 hours before use.
Cell Culture
[0079] PEGDM soft matrix ECM microarray slides were rinsed in DI
H.sub.2O for 1 hr followed by sterilization with 70% ethanol for
lhr prior to cell seeding. ECM microarray slides were equipped with
16 mm.times.16 mm silicon wells (Grace Bio-Labs) to partition
individual microarray replicates. Rat pulmonary arterial smooth
muscle cells (rPASMCs) were obtained from distal bovine vascular
arteries. The cells were maintained in Dulbecco's Modified Eagle's
Medium (Cellgo DMEM, Mediatech Inc, Manassas Va.), with 10% fetal
bovine serum (Gemini Bio-products, West Sacramento, Calif.) and 1%
Pen/Strep. Cell passages of 3-8 were used for all experiments.
Bovine Aortic Smooth Muscle Cells (BASMCs) were suspended at a
concentration of 10e6 cells per ml in serum free media. The cell
suspension was dispensed onto the ECM microarray within the gasket
region at a cell density of 10e5 cells per array and incubated for
2 hours. The arrays were then gently aspirated by submerging into a
large chamber filled with prewarmed media. Culture media was
changed daily.
[0080] Rat mesenchymal stem cells (rMSCs) with passages 2-5 were
cultured in Dulbeccos Modified Eagles Media, with 10% defined FBS
for MSCs and 1% Penn/Strep. rMSCs were suspended at a concentration
of 10e6 cells per ml in serum free media. The cell suspension was
dispensed onto the ECM microarray within the gasket region at a
cell density of 10e5 cells per array and incubated for 4 hours. The
arrays were then gently aspirated into a large chamber filled with
prewarmed media. Following aspiration, culture media (10% serum)
was introduced into the microarray wells. For cell cultures longer
than 24 hrs the culture media was changed daily.
Immunofluorescent Staining
[0081] Cell morphology was obtained by staining for cell nuclei
(DAPI) and cell cytoskeleton (phalloidin) after sample fixation in
4% formaldehyde. Fluorescently labeled cells were evaluated using
an epifluorescence microscope (Zeiss, Peabody, MA). Cell number and
morphology parameters were evaluated using Image? software.
Results
3.3.1 Nanofibrous Soft Matrix Preparation and Characterization
[0082] Nanofibrous hydrogels were prepared utilizing an
electrospinning technique whereby a photopolymerizable polymer is
spun onto TMSPSA functionalized glass surface, followed by UV
stabilization. The presence of methacrylate groups on the glass
surface allows for net stability and substrate longevity after
several rinses in aqueous solution, permitting unabridged function
for extended biological assays. PEGDM (MW 750) was selected for its
biocompatibility, ease of manipulation, elastic qualities and
commercial availability. The stabilization of PEGDM nets is
achieved via a radical chain photopolymerization between the
methacrylate groups in the presence of a photoinitiator and UV
light (352 nm). We employed mid-range FTIR to characterize the
degree of PEGDM conversion by monitoring the disappearance of the
reactive acrylate peak at 1637 cm.sup.-1 for samples over the
course of 15 minutes UV exposure. Results indicated that the
acrylate peak reduced by a maximum of 46% after 15 minutes of UV
exposure. The lack of efficient acrylate conversion is likely due
to the polymerization taking place in the dry state, reducing chain
mobility and active crosslinking domains for polymerization.
[0083] We investigated the nanofibrous architecture prepared from
electrospinning our PEGDM substrates using different microscopy
techniques in both the wet and dry states. Copolymerizing the PEGDM
nets with rhodamine-methacrylate permitted the visualization of
individual fiber diameter and geometry under confocal microscopy.
Employing scanning electron microscopy, we obtained high
magnification images of the prepared nanofibrous substrates in both
the dry and wet state. Both imaging methods indicate an approximate
fiber diameter after wetting of 0.5-1 .mu.m. Lack of beading or
webbing of the electrospun nets indicates optimal spinning
parameters with minimal artifacts.
[0084] To ascertain regulation of the elastic properties of the
prepared nanofibrous substrates, mechanical properties were
evaluated under shear using a parallel plate rheometer for PEGDM
specimens prepared under different UV exposures (2, 5, 10 and 15
minutes). A positive correlation between storage modulus and UV
exposure time was recorded for all specimens examined. Shear
modulus increased from 400 Pa to 15 kPa after 2 and 15 minutes UV
exposure respectively. The elastic properties measured are in
agreement with previous work reported by Wingate et al. using a
similar fabrication methodology (Wingate K, et., al., 2012.
Compressive elasticity of three dimensional nanofiber matrix
directs mesenchymal stem cell differentiation to vascular cells
with endothelial or smooth muscle cell markers. Acta Biomater 8,
1440).
Protein Microarray Design and Optimization
[0085] Array deposition of protein microdots produced repeatable
distinct microdots of average diameter 200 .mu.m and 500 .mu.m
pitch to pitch distance. We performed several print buffer
iterations using a control protein, albumin, to optimize dot
presentation, homogeneity and longevity upon nanofibrous PEGDM
substrates. Buffer glycerol content was found to influence the
printing parameters significantly. Glycerol content positively
correlated with dot circularity, whereas dot fluorescent intensity
correlated negatively to elevated glycerol content. A glycerol
content of 1% (v/v) was sufficient in retaining dot circularity
without reducing protein intensity markedly after substrate
incubation. Serial dilutions of control proteins albumin and
streptavidin revealed strong protein uptake by the nanofibrous
PEGDM hydrogels, with protein detected at as little as 15 .mu.g/ml
deposition concentration. A three dimensional protein microdot
presentation with approximate diameter of 200 .mu.m and substrate
penetration of 50 .mu.m was consistently achieved when printed upon
PEGDM nanofibrous substrates. Optimized print conditions are
amenable to global array deposition over large areas (10
mm.times.20 mm) with minimal perturbations of array organization
and layout.
[0086] We further assessed printing efficiency using several
extracellular matrix (ECM) proteins. Visual confirmation of
collagen I microdots was detectable at similar concentrations to
the control proteins (15-250 .mu.g/ml). We designed and printed a
combinatorial ECM microarray comprised of 6 ECM proteins, resulting
in 64 total conditions (rows) in replicates of eight (columns). We
confirmed protein retention for all ECM proteins through antigenic
immunostaining with collagen I and collagen IV immunofluorescence.
For all proteins investigated, immunofluorescence associated well
with expected distribution and intensity. Distinct
immunofluorescent detection of specific ECM proteins indicates
successful deposition of combinatorial designs.
Rat Mesenchymal Stem Cell Adhesion within 3D ECM Microarrays
[0087] Subsequent to the combinatorial ECM microarray design and
optimization, we proceeded to validate the biocompatibility and
selective attachment of stem cells on the ECM microarray
substrates. We seeded rat mesenchymal stem cells of late passage
(pll) to verify the selective attachment of cells to distinct
protein dots. We chose a late passage stem line for the preliminary
adhesion study as we found this provided improved adhesion to the
protein microdots, likely a result of a further differentiated stem
line. The rMSCs attached preferentially to the microarrayed regions
with little to no attachment observed on the pure PEGDM nanofibrous
substrate (FIG. 1A). Furthermore, upon closer inspection, several
of the distinct cellular islands represented three dimensional
neo-tissue microdomains of thickness approximately 50 .mu.m (FIG.
1B). Staining for cell nuclei identified distinct cell populations
associated with the ECM protein depositions. This attachment of
rMSCs to the ECM microarrays demonstrates the ability of stem cells
to selectively adhere to the microarrayed regions without dot to
dot communication.
[0088] To uncover the effects of substrate elasticity and ECM
combination on cellular adhesion, we evaluated the 24 hr. culture
of rMSCs on ECM microarrays prepared under two UV exposures, 5 and
15 minutes respectively. FIG. 2 depicts the unique adhesion of
rMSCs on stiff (15 min. UV)(top photo) and soft (5 min. UV)(bottom
photo) nanofibrous substrates. Cellular attachment and spreading
was confirmed for both elasticities, with improved spreading
detected for the softest condition (bottom photo).
[0089] To ascertain the effects of ECM composition on rMSC
attachment, cellular microarrays were stained for cell nuclei and
analyzed at 24 hrs. Each protein combination had detectable levels
of fluorescence with pure elastin representing the least favorable
adhesion point, and the elastin:fibronectin:collagen IV the most
favorable point for adhesion of rMSCs. Generally, protein dots
consisting of collagen IV or combined with other collagens (I and
III) lead to improved cellular attachment. Likewise, combinations
comprised mainly of laminin or elastin proved to have the least
adhesive strength.
Effect of ECM Composition on rMSCs Behavior and Fate Commitment
[0090] To explore the potential to modulate stem cell
differentiation events, we cultured rMSCs within our ECM
microarrays for 24 to 72 hours and subsequently monitored for the
early vascular marker PECAM. At 72-hour cell culture, rMSCs are
found to spread significantly within the ECM microdots, which, at
24 hours, were found to still be significantly round. The
expression of the early vascular marker was detectable at
significant intensities after 72-hour cell culture. Differentiation
capacity was found to be significantly affected by underlying
protein combination presented. The greatest expression was found
with protein dots comprised of fibronectin combined with the
collagens (I, III, IV), while the least was generally found with
combinations of elastin or laminin.
[0091] These data demonstrate that we developed a high throughput
method that allows for the rapid screening of a diversity of
engineered microenvironments with tunable matrix elasticity and
geometry, combined with specific ECM protein combination and/or
concentration.
Example 2
Preparation and Characterization of a Photoclickable Thiol-Ene
Poly(Ethylene Glycol) Hydrogel Cell Culture Matrix
[0092] We developed a microarray platform based on electrospun
nanofibrous photoclickable thiol-ene poly(ethylene glycol)
hydrogels. Thiol-ene polymerizations proceed by an orthogonal,
step-growth mechanism where one thiol reacts with one ene leading
to a highly homogenous distribution in crosslinks, thus imparting a
good control over substrate elasticity. Furthermore, it allows for
the subsequent modification of the already prepared electrospun
hydrogel substrates with ECM molecules such as peptides with high
reactivity and specificity.
Preparation and Characterization of Electrospun Thiol-Ene Hydrogel
Platform:
[0093] Four-arm Poly(ethylene glycol) norbornene (PEGNB; MW: 5 kDa)
was prepared as described elsewhere (Roberts J. J., Bryant S. J.,
Biomaterials 2013, 34(38), 9969-79). PEGNB (5-10 wt %, FIG. 3A),
polyethylene dithiol (1 kDa; thiol: ene=0.9), poly(ethylene oxide)
(3-7 wt %; MW: 400 kDa), and Irgacure 2959 (0.1 wt %) were
dissolved in DI water. Electrospun hydrogels were prepared by using
a custom set up using a 14-mm syringe at 30 kV. Needle-to-collector
distance (20-26 cm) and flow rate (0.4-1.2 ml/hr) were varied as
desired. Electrospun fibers were collected on a glass slide (25
mm.times.75 mm) previously modified with 3-(mercaptopropyl)
triethoxysilane. Substrates were subsequently exposed to UV (352 nm
light) with an average intensity of 5 mW/cm2 for specific time
points. Scanning electron microscopy was used to examine the
microstructure of the electrospun hydrogel substrates in both dry
and hydrated states. For hydrating, samples were soaked in
deionized water for 1 or 24 hours. Hydrated samples were shock
frozen in liquid nitrogen and lyophilized for 48 hours for SEM
imaging (FIG. 3B). Image J was used to measure fiber diameter.
Elastic properties of the electrospun hydrogels were characterized
using parallel plate rheometry.
Microarray Printing
[0094] Microarrays were prepared using a 2470 Aushon arrayer. A
printing buffer consisting of 1% glycerol and 0.2% Triton X-100 was
utilized for ALEXA FLUOR.TM. 488 or 546-05 maleimide printing.
Prepared microarrays were stored at 4.degree. C. in a humid
environment for 24 hours before confocal imaging.
Results
[0095] In this study, we developed electrospun hydrogel platform
using thiol-ene chemistry. The diameter of the electrospun
nanofibers ranged from 200-600 nm in the dry state. There was a 2
to 4-fold increase in fiber diameter when substrates were soaked
and imaged after lyophilization. The elastic modulus of the
substrates ranged from 1-5 kPa. Extracellular matrix (ECM) protein
molecules were deposited by combinatorial printing on these
electrospun hydrogel substrates. As a proof of concept, we have
demonstrated that maleimide dyes can be selectively printed with
high specificity. These studies indicate that this electrospun
hydrogel platform is highly tunable and we can create substrates
with different elastic properties by varying the molecular weight,
weight %, and thiol: ene ratio. Substrates with higher elastic
modulus (to cover the entire range of elasticity) can be prepared,
and ECM molecules relevant for stem cell differentiation can be
printed on these substrates. These data demonstrated that we have
developed a highly tunable platform with 3-D nanofibrous hydrogels
to facilitate high-throughput combinatorial screening of engineered
microenvironments for optimizing stem cell differentiation.
* * * * *